Laboratory of Norbert Perrimon, Ph.D.

Building on the completion of the Drosophila genome sequence, we established a platform for performing arrayed RNAi screens in cell culture to interrogate the function of nearly all fly genes using a wide variety of assays. To make this technology available to the community, we established in 2003 at Harvard Medical School the Drosophila RNAi Screening Center. Using the DRSC screening platform, the functions of the ~15,000 predicted Drosophila genes can be systematically analyzed to address questions in cell signaling, cell morphology, host-pathogen interactions, ion channel function, and many other topics. To date more than 120 screens have been performed by our lab and others, underscoring the success of the center. Over the years, we have added a number of screening reagents and developed bioinformatics tools to improve the platform. We renamed the center DRSC/TRiP-Functional Genomics Resources to better represent our current capabilities. Moreover, in 2019, we were funded to function as the Drosophila Research and Screening Center-Biomedical Technology Research Resource (DRSC-BTRR), which focuses on development of new screening and other technologies. We now have available for screening: genome-wide RNAi libraries and subset RNAi libraries (Kinase/Phosphatase; Ubiquitination; Transmembrane proteins; Transcription factors; RNA-binding proteins; Autophagy-related proteins; G-protein coupled receptors; Membrane-bound organelles; and Orthologs of human proteins for which there are FDA-approved drugs); Overexpression libraries (UAS-ORFs); and reagents for both gain-of-function (UAS-miR) and loss-of-function (UAS-miR-sponges) miRNA screens. Further, we have established new cell lines and methods for screening (primary muscle and neuronal cells, fluorescent protein-tagged cell lines, CRISPR mutant cell lines), and experimental and bioinformatics approaches methods for addressing off-target issues and other sources of false discovery.

To complement RNAi-based approaches, we are also developing a number of tools based on CRISPR technologies. We are using CRISPR to mutate or engineer cell lines that can be used for screening, and have developed efficient protocols in Drosophila cells, which are particularly challenging as they are polyploid and difficult to grow following single-cell isolation. CRISPR-generated mutant cell lines, in combination with RNAi, provide a robust platform for combinatorial screening. We have also generated stable nuclease-dead Cas9 activator (dCas9a) cell lines that can be used, after transfection with gRNAs, to perform overexpression screens - thus complementing loss of function screens. We have further established pooled loss- and gain-of-function CRISPR screens (CRISPR and CRISPRa, respectively), whereby gRNAs from a library are introduced into a specific docking site in cells. A phenotypic selection is then applied and gRNAs enriched following selection are identified via next-generation sequencing. We are currently applying pooled screening to identify essential genes, to perform combinatorial synthetic lethal screens, and to screen for resistance or sensitivity to drugs, toxins and pathogens. Finally, as an alternative to RNAi for partial loss-of-function screens using a Cas system, we have shown that Cas13 is an effective tool for CRISPRi in Drosophila cells. Advantages of CRISPRi as compared with RNAi are that the level of knockdown can be more tightly controlled and the system appears to have reduced off-target effects. Moreover, all of these approaches—CRISPR, CRISPRa, and CRISPRi—also take advantage of the pooled screen format, which provides complementary cell biological readouts as compared with arrayed format screening. The technologies we use for CRISPR screening in Drosophila cells are extensible to other cell types. One point of focus for DRSC-BTRR efforts is establishment of CRISPR pooled screening in cell lines from mosquito vectors of infectious diseases.

Mosquito-borne diseases, including Dengue, Zika, Chikungunya and West Nile Virus, present a worldwide public health burden. Mosquito cell lines exist that are able to become infected with viruses and are susceptible to mosquitocidal toxins, parasites, and drugs, but tools to allow deeper understanding of pathogen interactions with mosquito cells are lacking. We previously developed a recombination-mediated cassette exchange (RMCE) system that enables pooled CRISPR screening in Drosophila cells, as well as bioinformatics tools for identifying sgRNA designs for mosquitos and mosquito cell lines. We are now adapting the pooled-format CRISPR screening approach for use in cell lines from medically relevant mosquito species. With this advance, we hope to enable the mosquito community to easily perform genome-wide loss- or gain-of-function screens in cells, for example to discover entry and infection mechanisms of pathogens and drugs, as well as to aid in the functional annotation of mosquito genomes.

Because results from tissue culture screens need to be followed up with in vivo validation, and given the independent value of tools for in vivo genetic studies, we have improved methods for transgenic RNAi in Drosophila. We demonstrated that shRNAs are more efficient and specific than long dsRNA when expressed as transgenes, and generated a genome scale collection of >13,000 lines covering 75% of the genome in our optimized VALIUM vectors (85% of highly conserved genes). This collection is available to the community through the Bloomington Drosophila Stock Center. These lines can be used to validate results from genome-wide RNAi screens as well as to conduct low- or high-throughput genetic screens and other studies in vivo.

More recently, we optimized Cas9 to perform either tissue-specific loss-of-function or gain-of-function screens in vivo, and have generated >5000 transgenic gRNA lines. For gain-of-function, the lines express gRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain. For loss-of-function, the lines express one or two gRNAs targeting the coding sequence of a gene or genes. These lines can be combined with tissue-specific delivery of Cas9 to generate clones of cells (mosaics) or combined with germline expression of Cas9 to generate null mutations. In addition, these gRNA fly stock resources can be used for genome engineering of the promoter or coding regions.

We continue to evaluate and create new methods for genome engineering. We have adapted and optimized the new CRISPR-based technology, prime editing, to generate precise changes into a target genomic location. In a collaboration with Sebastian Kadener’s lab at Brandeis, we generated a new set of PspCas13b and RfxCas13d expression constructs that can be used to target RNA in cells and in vivo. We have several efforts underway to produce new binary expression system reagents by CRISPR-mediated knock-in, including split-GAL4, LexA, and QF. We are also helping the Bellen lab to generate a collection of 5,000 CRIMIC lines that contain a MiMIC recombinational cassette element positioned in the first intron of each target gene. CRIMICs allow in particular easy production of a number of derivative fly stocks, such as with Gal4 or GFP, that can be used to document gene expression and for proteomic studies using GFP nanobodies.

Proximity Labeling: Characterizing the proteome composition of organelles and subcellular regions of living cells can facilitate the understanding of cellular organization as well as protein interactome networks. Proximity labeling-based methods coupled with mass spectrometry (MS) offer a high-throughput approach for systematic analysis of spatially-restricted proteomes. Proximity labeling utilizes enzymes that generate reactive radicals to covalently tag neighboring proteins. The tagged endogenous proteins can then be isolated for further analysis by MS. To analyze protein-protein interactions or identify components that localize to discrete subcellular compartments, we developed tools based on APEX, an engineered ascorbate peroxidase derived from plants,  and  BioID, a mutant form of the biotin ligase BirA from E coli and demonstrated their use  for in vivo studies. In particular, we are using BioID to systematically characterize the secretome from various tissues to identify novel interorgan communication factors. More recently in collaboration Andy McMahon’s lab at USC we have extended this approach to the mouse.

NanoTags: One of the key reagents to understand protein expression and function are antibodies. Antibodies allow protein visualization by immunostaining, biochemical study by immunoprecipitation and western blot, and proteomic study by IP-MS. Despite the importance of antibodies in biology study, antibodies are not available for most fly proteins.  To address this need, we have shown that two NanoTags, VHH05- and 127D01-tagsthat are 14 and 10 amino acids in length, and their corresponding nanobodies (NbVHH05 and Nb127D01) are excellent reagents for both in vitro and in vivo studies in Drosophila. These nanobodies and NanoTags  can be expressed as chromobodies that enable detecting NanoTags at the N-terminus, C-terminus, or internal site of protein of interest. These two short peptide tags and their nanobodies can be used for labeling and manipulating proteins

We have developed a series of bioinformatics tools that provide the research community with well-designed, user-friendly resources that impact research at all stages, from project design to data analysis and integration. These tools, which are available via DRSC/TRiP Functional Genomics Resources, include:

Our most popular tools are: DIOPT, an integrative tool for ortholog predictions among major model organisms. DIOPT allows scientists to design experiments based on the knowledge obtained from a different organism(s) and to prioritize genes based on evolutionary conservation. DIOPT has been well received by the scientific community and has been integrated into FlyBase, PomBase, MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) and AGR (Alliance of Genome Resources); Gene2Function integrates annotation information for orthologs of multiple species in unified interfaces; RSVP allows scientists to mine the validation and phenotype data for in vivo RNAi and sgRNA stocks; MIST (Molecular Interaction Search Tool) for mining, visualization and analysis of network data, iProteinDB for mining protein post-translational modification (PTM) data and comparison of PTM data across species and DRscDB for mining and comparison of single-cell RNA-seq data across species.